A time-resolved single-cell roadmap of the logic driving anterior neural crest diversification from neural border to migration stages.

Neural crest cells exemplify cellular diversification from a multipotent progenitor population. However, the full sequence of early molecular choices orchestrating the emergence of neural crest heterogeneity from the embryonic ectoderm remains elusive. Gene-regulatory-networks (GRN) govern early development and cell specification toward definitive neural crest. Here, we combine ultradense single-cell transcriptomes with machine-learning and large-scale transcriptomic and epigenomic experimental validation of selected trajectories, to provide the general principles and highlight specific features of the GRN underlying neural crest fate diversification from induction to early migration stages using Xenopus frog embryos as a model. During gastrulation, a transient neural border zone state precedes the choice between neural crest and placodes which includes multiple converging gene programs. During neurulation, transcription factor connectome, and bifurcation analyses demonstrate the early emergence of neural crest fates at the neural plate stage, alongside an unbiased multipotent-like lineage persisting until epithelial-mesenchymal transition stage. We also decipher circuits driving cranial and vagal neural crest formation and provide a broadly applicable high-throughput validation strategy for investigating single-cell transcriptomes in vertebrate GRNs in development, evolution, and disease.

Human neural tube morphogenesis in vitro by geometric constraints.

Understanding human organ formation is a scientific challenge with far-reaching medical implications1,2. Three-dimensional stem-cell cultures have provided insights into human cell differentiation3,4. However, current approaches use scaffold-free stem-cell aggregates, which develop non-reproducible tissue shapes and variable cell-fate patterns. This limits their capacity to recapitulate organ formation. Here we present a chip-based culture system that enables self-organization of micropatterned stem cells into precise three-dimensional cell-fate patterns and organ shapes. We use this system to recreate neural tube folding from human stem cells in a dish. Upon neural induction5,6, neural ectoderm folds into a millimetre-long neural tube covered with non-neural ectoderm. Folding occurs at 90% fidelity, and anatomically resembles the developing human neural tube. We find that neural and non-neural ectoderm are necessary and sufficient for folding morphogenesis. We identify two mechanisms drive folding: (1) apical contraction of neural ectoderm, and (2) basal adhesion mediated via extracellular matrix synthesis by non-neural ectoderm. Targeting these two mechanisms using drugs leads to morphological defects similar to neural tube defects. Finally, we show that neural tissue width determines neural tube shape, suggesting that morphology along the anterior-posterior axis depends on neural ectoderm geometry in addition to molecular gradients7. Our approach provides a new route to the study of human organ morphogenesis in health and disease.

The complete cell lineage and MAPK- and Otx-dependent specification of the dopaminergic cells in the Ciona brain.

The Ciona larva has served as a unique model for understanding the development of dopaminergic cells at single-cell resolution owing to the exceptionally small number of neurons in its brain and its fixed cell lineage during embryogenesis. A recent study suggested that the transcription factors Fer2 and Meis directly regulate the dopamine synthesis genes in Ciona, but the dopaminergic cell lineage and the gene regulatory networks that control the development of dopaminergic cells have not been fully elucidated. Here, we reveal that the dopaminergic cells in Ciona are derived from a bilateral pair of cells called a9.37 cells at the center of the neural plate. The a9.37 cells divide along the anterior-posterior axis, and all of the descendants of the posterior daughter cells differentiate into the dopaminergic cells. We show that the MAPK pathway and the transcription factor Otx are required for the expression of Fer2 in the dopaminergic cell lineage. Our findings establish the cellular and molecular framework for fully understanding the commitment to dopaminergic cells in the simple chordate brain.

Scribble mutation disrupts convergent extension and apical constriction during mammalian neural tube closure.

Morphogenesis of the vertebrate neural tube occurs by elongation and bending of the neural plate, tissue shape changes that are driven at the cellular level by polarized cell intercalation and cell shape changes, notably apical constriction and cell wedging. Coordinated cell intercalation, apical constriction, and wedging undoubtedly require complex underlying cytoskeletal dynamics and remodeling of adhesions. Mutations of the gene encoding Scribble result in neural tube defects in mice, however the cellular and molecular mechanisms by which Scrib regulates neural cell behavior remain unknown. Analysis of Scribble mutants revealed defects in neural tissue shape changes, and live cell imaging of mouse embryos showed that the Scrib mutation results in defects in polarized cell intercalation, particularly in rosette resolution, and failure of both cell apical constriction and cell wedging. Scrib mutant embryos displayed aberrant expression of the junctional proteins ZO-1, Par3, Par6, E- and N-cadherins, and the cytoskeletal proteins actin and myosin. These findings show that Scribble has a central role in organizing the molecular complexes regulating the morphomechanical neural cell behaviors underlying vertebrate neurulation, and they advance our understanding of the molecular mechanisms involved in mammalian neural tube closure.

Surface contraction waves or cell proliferation waves in the presumptive neurectoderm during amphibian gastrulation: Mexican axolotl versus African clawed frog.

This essay represents a critical analysis of the literary data on various types of waves occurring in the amphibian embryos during gastrulation. A surface contraction wave travels through the presumptive neurectoderm during Mexican axolotl gastrulation. This wave coincides temporally and spatially with involution of the inducing chordomesoderm and with the prospective neural plate. By contrast, there is no similar surface contraction wave during African clawed frog gastrulation. However, the clawed frog displays the waves of DNA synthesis and mitosis in the presumptive neurectoderm during gastrulation, whereas no such waves were discovered in axolotl gastrulae. These sets of experimental data are in accordance with the contemporary concept of considerable ontogenetic diversity of the class Amphibia.

STRAP regulates alternative splicing fidelity during lineage commitment of mouse embryonic stem cells.

Alternative splicing (AS) is involved in cell fate decisions and embryonic development. However, regulation of these processes is poorly understood. Here, we have identified the serine threonine kinase receptor-associated protein (STRAP) as a putative spliceosome-associated factor. Upon Strap deletion, there are numerous AS events observed in mouse embryoid bodies (EBs) undergoing a neuroectoderm-like state. Global mapping of STRAP-RNA binding in mouse embryos by enhanced-CLIP sequencing (eCLIP-seq) reveals that STRAP preferably targets transcripts for nervous system development and regulates AS through preferred binding positions, as demonstrated for two neuronal-specific genes, Nnat and Mark3. We have found that STRAP involves in the assembly of 17S U2 snRNP proteins. Moreover, in Xenopus, loss of Strap leads to impeded lineage differentiation in embryos, delayed neural tube closure, and altered exon skipping. Collectively, our findings reveal a previously unknown function of STRAP in mediating the splicing networks of lineage commitment, alteration of which may be involved in early embryonic lethality in mice.

Mechanisms of melanocyte polarity and differentiation: What can we learn from other neuroectoderm-derived lineages?

Melanocytes are neuroectoderm-derived pigment-producing cells with highly polarized dendritic morphology. They protect the skin against ultraviolet radiation by providing melanin to neighbouring keratinocytes. However, the mechanisms underlying melanocyte polarization and its relevance for diseases remain mostly elusive. Numerous studies have instead revealed roles for polarity regulators in other neuroectoderm-derived lineages including different neuronal cell types. Considering the shared ontogeny and morphological similarities, these lineages may be used as reference models for the exploration of melanocyte polarity, for example, regarding dendrite formation, spine morphogenesis and polarized organelle transport. In this review, we summarize and compare the latest progress in understanding polarity regulation in neuronal cells and melanocytes and project key open questions for future work.

Recapitulation of Neural Crest Specification and EMT via Induction from Neural Plate Border-like Cells.

Neural crest cells (NCCs) contribute to several tissues during embryonic development. NCC formation depends on activation of tightly regulated molecular programs at the neural plate border (NPB) region, which initiate NCC specification and epithelial-to-mesenchymal transition (EMT). Although several approaches to investigate NCCs have been devised, these early events of NCC formation remain largely unknown in humans, and currently available cellular models have not investigated EMT. Here, we report that the E6 neural induction protocol converts human induced pluripotent stem cells into NPB-like cells (NBCs), from which NCCs can be efficiently derived. NBC-to-NCC induction recapitulates gene expression dynamics associated with NCC specification and EMT, including downregulation of NPB factors and upregulation of NCC specifiers, coupled with other EMT-associated cell-state changes, such as cadherin modulation and activation of TWIST1 and other EMT inducers. This strategy will be useful in future basic or translational research focusing on these early steps of NCC formation.

Investigating cellular and molecular mechanisms of neurogenesis in Capitella teleta sheds light on the ancestor of Annelida.

BACKGROUND: Diverse architectures of nervous systems (NSs) such as a plexus in cnidarians or a more centralized nervous system (CNS) in insects and vertebrates are present across Metazoa, but it is unclear what selection pressures drove evolution and diversification of NSs. One underlying aspect of this diversity lies in the cellular and molecular mechanisms driving neurogenesis, i.e. generation of neurons from neural precursor cells (NPCs). In cnidarians, vertebrates, and arthropods, homologs of SoxB and bHLH proneural genes control different steps of neurogenesis, suggesting that some neurogenic mechanisms may be conserved. However, data are lacking for spiralian taxa. RESULTS: To that end, we characterized NPCs and their daughters at different stages of neurogenesis in the spiralian annelid Capitella teleta. We assessed cellular division patterns in the neuroectoderm using static and pulse-chase labeling with thymidine analogs (EdU and BrdU), which enabled identification of NPCs that underwent multiple rounds of division. Actively-dividing brain NPCs were found to be apically-localized, whereas actively-dividing NPCs for the ventral nerve cord (VNC) were found apically, basally, and closer to the ventral midline. We used lineage tracing to characterize the changing boundary of the trunk neuroectoderm. Finally, to start to generate a genetic hierarchy, we performed double-fluorescent in-situ hybridization (FISH) and single-FISH plus EdU labeling for neurogenic gene homologs. In the brain and VNC, Ct-soxB1 and Ct-neurogenin were expressed in a large proportion of apically-localized, EdU+ NPCs. In contrast, Ct-ash1 was expressed in a small subset of apically-localized, EdU+ NPCs and subsurface, EdU- cells, but not in Ct-neuroD+ or Ct-elav1+ cells, which also were subsurface. CONCLUSIONS: Our data suggest a putative genetic hierarchy with Ct-soxB1 and Ct-neurogenin at the top, followed by Ct-ash1, then Ct-neuroD, and finally Ct-elav1. Comparison of our data with that from Platynereis dumerilii revealed expression of neurogenin homologs in proliferating NPCs in annelids, which appears different than the expression of vertebrate neurogenin homologs in cells that are exiting the cell cycle. Furthermore, differences between neurogenesis in the head versus trunk of C. teleta suggest that these two tissues may be independent developmental modules, possibly with differing evolutionary trajectories.

Profiling of O-acetylated Gangliosides Expressed in Neuroectoderm Derived Cells.

The expression and biological functions of oncofetal markers GD2 and GD3 were extensively studied in neuroectoderm-derived cancers in order to characterize their potential as therapeutic targets. Using immunological approaches, we previously identified GD3, GD2, and OAcGD2 expression in breast cancer (BC) cell lines. However, antibodies specific for O-acetylated gangliosides are not exempt of limitations, as they only provide information on the expression of a limited set of O-acetylated ganglioside species. Consequently, the aim of the present study was to use structural approaches in order to apprehend ganglioside diversity in melanoma, neuroblastoma, and breast cancer cells, focusing on O-acetylated species that are usually lost under alkaline conditions and require specific analytical procedures. We used purification and extraction methods that preserve the O-acetyl modification for the analysis of native gangliosides by MALDI-TOF. We identified the expression of GM1, GM2, GM3, GD2, GD3, GT2, and GT3 in SK-Mel28 (melanoma), LAN-1 (neuroblastoma), Hs 578T, SUM 159PT, MDA-MB-231, MCF-7 (BC), and BC cell lines over-expressing GD3 synthase. Among O-acetylated gangliosides, we characterized the expression of OAcGM1, OAcGD3, OAcGD2, OAcGT2, and OAcGT3. Furthermore, the experimental procedure allowed us to clearly identify the position of the sialic acid residue that carries the O-acetyl group on b- and c-series gangliosides by MS/MS fragmentation. These results show that ganglioside O-acetylation occurs on both inner and terminal sialic acid residue in a cell type-dependent manner, suggesting different O-acetylation pathways for gangliosides. They also highlight the limitation of immuno-detection for the complete identification of O-acetylated ganglioside profiles in cancer cells.

Pineal progenitors originate from a non-neural territory limited by FGF signalling.

The embryonic development of the pineal organ, a neuroendocrine gland on top of the diencephalon, remains enigmatic. Classic fate-mapping studies suggested that pineal progenitors originate from the lateral border of the anterior neural plate. We show here, using gene expression and fate mapping/lineage tracing in zebrafish, that pineal progenitors originate, at least in part, from the non-neural ectoderm. Gene expression in chick indicates that this non-neural origin of pineal progenitors is conserved in amniotes. Genetic repression of placodal, but not neural crest, cell fate results in pineal hypoplasia in zebrafish, while mis-expression of transcription factors known to specify placodal identity during gastrulation promotes the formation of ectopic pineal progenitors. We also demonstrate that fibroblast growth factors (FGFs) position the pineal progenitor domain within the non-neural border by repressing pineal fate and that the Otx transcription factors promote pinealogenesis by inhibiting this FGF activity. The non-neural origin of the pineal organ reveals an underlying similarity in the formation of the pineal and pituitary glands, and suggests that all CNS neuroendocrine organs may require a non-neural contribution to form neurosecretory cells.

Developmental analyses of mouse embryos and adults using a non-overlapping tracing system for all three germ layers.

Genetic lineage-tracing techniques are powerful tools for studying specific cell populations in development and pathogenesis. Previous techniques have mainly involved systems for tracing a single gene, which are limited in their ability to facilitate direct comparisons of the contributions of different cell lineages. We have developed a new combinatorial system for tracing all three germ layers using self-cleaving 2A peptides and multiple site-specific recombinases (SSRs). In the resulting TRiCK (TRiple Coloured germ layer Knock-in) mice, the three germ layers are conditionally and simultaneously labelled with distinct fluorescent proteins via embryogenesis. We show that previously reported ectopic expressions of lineage markers are the outcome of secondary gene expression. The results presented here also indicate that the commitment of caudal axial stem cells to neural or mesodermal fate proceeds without lineage fluctuations, contrary to the notion of their bi-potency. Moreover, we developed IMES, an optimized tissue clearing method that is highly compatible with a variety of fluorescent proteins and immunostaining, and the combined use of TRiCK mice and IMES can facilitate comprehensive analyses of dynamic contributions of all three germ layers.

Single-cell qPCR demonstrates that Repsox treatment changes cell fate from endoderm to neuroectoderm and disrupts epithelial-mesenchymal transition.

A definitive endodermal cell lineage is a prerequisite for the efficient generation of mature endoderm derivatives that give rise to organs, such as the pancreas and liver. We previously reported that the induction of mesenchymal definitive endoderm cells depends on autocrine TGF-ß signaling and that pharmacological blockage of TGF-ß signaling by Repsox disrupts endoderm specification. The definitive endoderm arises from a primitive streak, which depends largely on TGF-ß signaling. If the TGF-ß pathway is blocked by Repsox, cell fate after the primitive streak induction is so-far unknown. We report here, that an induced primitive streak cell-population contained many T/SOX2 co-expressing cells, and subsequent inhibition of TGF-ß signaling by Repsox promoted neuroectodermal cell fate, which was characterized using single-cell qPCR analysis and immunostaining. The process of epithelial-to-mesenchymal transition, which is inherent to the process of definitive endoderm differentiation, was also disrupted upon Repsox treatment. Our findings may provide a new approach to produce neural progenitors.

Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions.

SOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. How temporal fluctuations in their expression levels bias lineage commitment is unknown. Here, we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cells towards both neuroectodermal and mesendodermal fates in undirected differentiation. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

Embryoid Body Differentiation of Mouse Embryonic Stem Cells into Neurectoderm and Neural Progenitors.

Mouse embryonic stem cells (mESCs) are pluripotent cells capable of differentiating in vitro to form the ~200 types of cells of the developing embryo and adult, including cells of the nervous system. This makes mESCs a useful tool for studying the molecular mechanisms of mammalian embryonic development. Many protocols involving the use of growth factors and small molecules to differentiate mESCs into neural progenitors and neurons currently exist. However, there is a paucity of protocols available that recapitulate the developmental process. Our laboratory has developed a protocol to recapitulate mammalian neural lineage development by differentiating mESCs to mature neurons via intermediate cell populations observed during in vivo embryo development. This protocol uses the amino acid L-proline to direct the differentiation of mESCs, grown as embryoid bodies, into Sox1+ neurectoderm, followed by differentiation to form Nestin+, BLBP+, and NeuN+ neural cell types.

Gain of 20q11.21 in Human Pluripotent Stem Cells Impairs TGF-ß-Dependent Neuroectodermal Commitment.

Gain of 20q11.21 is one of the most common recurrent genomic aberrations in human pluripotent stem cells. Although it is known that overexpression of the antiapoptotic gene Bcl-xL confers a survival advantage to the abnormal cells, their differentiation capacity has not been fully investigated. RNA sequencing of mutant and control hESC lines, and a line transgenically overexpressing Bcl-xL, shows that overexpression of Bcl-xL is sufficient to cause most transcriptional changes induced by the gain of 20q11.21. Moreover, the differentially expressed genes in mutant and Bcl-xL overexpressing lines are enriched for genes involved in TGF-ß- and SMAD-mediated signaling, and neuron differentiation. Finally, we show that this altered signaling has a dramatic negative effect on neuroectodermal differentiation, while the cells maintain their ability to differentiate to mesendoderm derivatives. These findings stress the importance of thorough genetic testing of the lines before their use in research or the clinic.

Whsc1 links pluripotency exit with mesendoderm specification.

How pluripotent stem cells differentiate into the main germ layers is a key question of developmental biology. Here, we show that the chromatin-related factor Whsc1 (also known as Nsd2 and MMSET) has a dual role in pluripotency exit and germ layer specification of embryonic stem cells. On induction of differentiation, a proportion of Whsc1-depleted embryonic stem cells remain entrapped in a pluripotent state and fail to form mesendoderm, although they are still capable of generating neuroectoderm. These functions of Whsc1 are independent of its methyltransferase activity. Whsc1 binds to enhancers of the mesendodermal regulators Gata4, T (Brachyury), Gata6 and Foxa2, together with Brd4, and activates the expression of these genes. Depleting each of these regulators also delays pluripotency exit, suggesting that they mediate the effects observed with Whsc1. Our data indicate that Whsc1 links silencing of the pluripotency regulatory network with activation of mesendoderm lineages.

[Neural Crest-An Unusual Population of Embryonic Cells].

The neural crest (NC) in embryos of vertebrates represents a cell population formed at the border of the neural plate. These cells retain pluripotency, express a set of specific markers, and become multipotent upon their migration away from the neural tube to give rise to numerous derivatives. The genes specific for vertebrate NC appeared in evolution long before vertebrates. Abnormal development of NC cells causes numerous pathologies in humans.

In Vivo and In Vitro Quantitative Analysis of Neural Crest Cell Migration.

The neural crest is an embryonic cell population induced at the border of the neural plate from where it delaminates and migrates long distances across the embryo. Due to its extraordinary migratory capabilities, the neural crest has become a powerful system to study cellular and molecular aspects of collective and single cell migration both in vivo and in vitro. Here we provide detailed protocols used to perform quantitative analysis of molecular and cellular aspects of Xenopus laevis neural crest cell migration, both in vivo and in vitro.

GPCR-independent activation of G proteins promotes apical cell constriction in vivo.

Heterotrimeric G proteins are signaling switches that control organismal morphogenesis across metazoans. In invertebrates, specific GPCRs instruct G proteins to promote collective apical cell constriction in the context of epithelial tissue morphogenesis. In contrast, tissue-specific factors that instruct G proteins during analogous processes in vertebrates are largely unknown. Here, we show that DAPLE, a non-GPCR protein linked to human neurodevelopmental disorders, is expressed specifically in the neural plate of Xenopus laevis embryos to trigger a G protein signaling pathway that promotes apical cell constriction during neurulation. DAPLE localizes to apical cell-cell junctions in the neuroepithelium, where it activates G protein signaling to drive actomyosin-dependent apical constriction and subsequent bending of the neural plate. This function is mediated by a Gα-binding-and-activating (GBA) motif that was acquired by DAPLE in vertebrates during evolution. These findings reveal that regulation of tissue remodeling during vertebrate development can be driven by an unconventional mechanism of heterotrimeric G protein activation that operates in lieu of GPCRs.